Method of screening physiologically active substance

Abstract

The present invention provides a method of screening a biologically active substance or a candidate substance for a biologically active substance, possessing an activity for controlling gene expression to the same extent to that of DGE; a gene controlling agent which is effective for maintaining homeostasis in a living body, for instance, purposed for mitigation or prevention of stress; and a method of controlling a gene.

1 . A method of screening a biologically active substance, characterized in that the method comprises the steps of: 
 (1) loading to a living body either 1) a test substance or 2) a compound of the following (i): 
 (i) at least one compound selected from the group consisting of a compound represented by the following formula (I):  
                     
  wherein X and Y are H or CH 2 OH, with proviso that when X is CH 2 OH, Y is H,  
  and when X is H, Y is CH 2 OH,  
  a derivative thereof and a salt thereof; and/or  
  a compound of the following (ii):  
 (ii) at least one compound selected from the group consisting of 3,6-anhydrogalactose represented by the following formula (II):  
                     
  an aldehyde thereof, a hydrate thereof, and a 2-O-methylated product and a 2-O-sulfated product of the foregoing compounds; and/or at least one compound selected from the group consisting of soluble saccharide compounds having said compound at reducing end;  
   (2) determining an expression level of a gene in the living body of said (1), and comparing resulting values between a case where 1) is loaded and a case where 2) is loaded; and    (3) selecting a test substance which causes a change in an expression level of at least one gene to substantially the same extent as a change where 2) is loaded.    
     
     
         2 . A method of screening a biologically active substance, characterized in that the method comprises the steps of: 
 (1) loading a test substance to a living body;    (2) determining an expression level of a gene in the living body of said (1) and an expression level of a gene in the living body without the loading, and comparing resulting values therebetween; and    (3) selecting a test substance which decreases an expression level of at least one gene selected from the following Group A, Group C, Group E and Group F, and/or increases an expression level of at least one gene selected from the following Group B, Group D and Group G: 
 Group A: 
 Bone morphogenetic protein-6 [BMP-6 (GenBank Accession No. NM — 001718)] gene,  
 Chemokine receptor-1 [CCR-1 (GenBank Accession No. NM — 001295)] gene,  
 Hepatocyte growth factor [HGF (GenBank Accession No. X16323)] gene,  
 Intercellular adhesion molecule-1 [ICAM-1 (GenBank Accession No. NM — 000201)] gene,  
 Interleukin-1β [IL-1β (GenBank Accession No. X02532)] gene,  
 Interleukin-16 [IL-16 (GenBank Accession No. M90391)] gene,  
 Leukemia inhibitory factor [LIF (GenBank Accession No. NM — 002309)] gene,  
 Osteopontin [Osteopontin (GenBank Accession No. NM — 000582)] gene,  
 Tumor necrosis factor-α [TNF-α (GenBank Accession No. X02910)] gene,  
 Neuropilin-1 [Neuropilin-1 (GenBank Accession No. NM — 003873)] gene,  
 signal transducer and activator of transcription 6 [STAT6 (GenBank Accession No. AF067575)] gene, and  
 Gene containing  Homo Sapiens  cDNA: FLJ22298 fis, clone HRC04637 (GenBank Accession No. AK025951),  
 
 Group B: 
 Heme oxygenase-1 [HO-1 (GenBank Accession No. NM — 002133)] gene,  
 Thioredoxin reductase-1 (GenBank Accession No. AF106697) gene,  
 putative translation initiation factor [SUIL (GenBank Accession No. AF083441)] gene, and  
   Homo sapiens  hypothetical protein [HSPCO14 (GenBank Accession No. NM — 015932)] gene;  
 
 Group C: 
 N-myc downstream regulated gene 1 [NDRG1 (GenBank Accession No. NM — 006096)] gene,  
 adenosine deaminase, RNA specific [ADAR (GenBank Accession No. NM — 015841)] gene,  
 splicing factor 3b, subunit2 [SF3B2 (GenBank Accession No. NM — 006842)] gene,  
 interleukin enhancer binding factor 3 [ILF3 (GenBank Accession No. NM — 004516)] gene,  
 valyl-tRNA synthetase 2 [VARS2 (GenBank Accession No. NM — 006295)] gene, and  
 Gene containing the nucleotide sequence shown in SEQ ID NO: 12 of Sequence Listing;  
 
 Group D: 
 eukaryotic translation elongation factor 1 alpha 1 [EEF1A1 (GenBank Accession No. NM — 001402)] gene,  
 ribosomal protein L17 [RPL17 (GenBank Accession No. NM — 000985)] gene,  
 ribosomal protein L24 [RPL24 (GenBank Accession No. NM — 000986)] gene,  
 splicing factor, arginine/serine-rich 2 [SFRS2 (GenBank Accession No. NM — 003016)] gene,  
 tumor protein, translationally-controlled 1 [TPT1 (GenBank Accession No. NM — 003295)] gene,  
 ubiquinol-cytochrome c reductase binding protein [UQCRB (GenBank Accession No. NM — 006294)] gene,  
 ribosomal protein S15a [RPS15A (GenBank Accession No. NM — 001019)] gene, and  
 ribosomal protein L27 [RPL27 (GenBank Accession No. NM — 000988)] gene;  
 
 Group E: 
 Interleukin-2 receptor α [IL2RA (GenBank Accession No. X01057)] gene,  
 Interleukin-6 [IL6 (GenBank Accession No. X04430)] gene,  
 CD166 (GenBank Accession No. Y10183) gene,  
 Interferon regulatory factor-1 [IRF1 (GenBank Accession No. X14454)] gene,  
 Interleukin-15 receptor α [IL1SRA (GenBank Accession No. U31628)] gene,  
 Nuclear factor kappa-b p105 Subunit [NFKB1 (GenBank Accession No. M58603)] gene,  
 Caspase-1 [CASP1 (GenBank Accession No. M87507)] gene,  
 Interleukin-1β [IL1B (GenBank Accession No. M15330)] gene,  
 Tumor necrosis factor-α [TNF-α (GenBank Accession No. X02910)] gene,  
 Gro1 oncogene [GRO1 (GenBank Accession No. X54489)] gene,  
 Interleukin-1α [IL1A (GenBank Accession No. M28983)] gene,  
 Inhibin βA [INHBA (GenBank Accession No. J03634)] gene,  
 Interleukin-7 receptor [IL7R (GenBank Accession No. M29696)] gene,  
 Precursor B cell colony enhancing factor [PBEF (GenBank Accession No. U02020)] gene,  
 Liver and activation regulated chemokine [LARC (GenBank Accession No. U64197)] gene, and  
 Interleukin-8 [IL8 (GenBank Accession No. M26383)] gene;  
 
 Group F: 
 Colony-stimulating factor-1 receptor [CSF1R (GenBank Accession No. X03663)] gene,  
 Chemokine (C-X-C motif) receptor-4 [CXCR4 (GenBank Accession No. AF147204)] gene, and  
 Endoglin [ENG (GenBank Accession No. NM — 000118)] gene; and  
 
 Group G: 
 Heme oxygenase-1 [HMOX1 (GenBank Accession No. Z82244)] gene.  
 
   
     
     
         3 . A method of screening a biologically active substance, characterized in that the method comprises the steps of: 
 (1) loading stress to a living body, and loading a test substance with stress to a living body, respectively;    (2) determining each of expression levels of a gene in the living body of said (1) and in a normal living body without any of the loading; and    (3) comparing the expression levels of a gene obtained in said (2) between the living body of said (1) and the normal living body, and selecting a test substance showing at least one gene expression pattern selected from the gene expression patterns of the followings (a) to (g): 
 (a) an expression level of at least one gene selected from Group A as defined in  claim 2  increases when stress is loaded to a living body, and decreases when a test substance and stress are loaded to a living body, as compared to that of the normal living body;  
 (b) an expression level of at least one gene selected from Group B as defined in  claim 2  increases when stress is loaded to a living body, and further increases when a test substance and stress are loaded to a living body, as compared to that of the normal living body;  
   (c) an expression level of at least one gene selected from Group C as defined in  claim 2  decreases when stress is loaded to a living body, and further decreases when a test substance and stress are loaded to a living body, as compared to that of the normal living body;    (d) an expression level of at least one gene selected from Group D as defined in  claim 2  decreases when stress is loaded to a living body, and increases when a test substance and stress are loaded to a living body, as compared to that of the normal living body;    (e) an expression level of at least one gene selected from Group E as defined in  claim 2  increases when stress is loaded to a living body, and decreases when a test substance and stress are loaded to a living body, as compared to that of the normal living body;    (f) an expression level of at least one gene selected from Group F as defined in  claim 2  is the same extent as that of the normal living body when stress is loaded to a living body, and decreases when a test substance and stress are loaded to a living body, as compared to that of the normal living body; and    (g) an expression level of at least one gene selected from Group G as defined in  claim 2  is the same extent as that of the normal living body when stress is loaded to a living body, and increases when a test substance and stress are loaded to a living body, as compared to that of the normal living body.    
     
     
         4 . The method according to  claim 3 , wherein the stress is a treatment with phorbol myristate acetate, and wherein the gene expression pattern is selected from the gene expression patterns of said (a) to (d).  
     
     
         5 . The method according to  claim 3 , wherein the stress is a treatment with lipopolysaccharide, and wherein the gene expression pattern is selected from the gene expression patterns of said (e) to (g).  
     
     
         6 . The method according to any one of  claims 1  to  5 , wherein the living body is an organism individual or a cultured cell.  
     
     
         7 . The method according to any one of  claims 1  to  6 , wherein the expression level of a gene is determined by an amount of mRNA transcribed from the gene.  
     
     
         8 . The method according to  claim 7 , wherein the expression level of a gene is determined by hybridization method using a DNA array.  
     
     
         9 . A DNA array usable for the method of  claim 8 , characterized in that at least two genes selected from the genes of Groups A to G as defined in  claim 2  or fragments thereof are immobilized on a support at a given position.  
     
     
         10 . A gene controlling agent characterized in that the gene controlling agent comprises a compound possessing an action of decreasing an expression level of at least one gene selected from Group A, Group C, Group E and Group F as defined in  claim 2 , and/or increasing an expression level of at least one gene selected from Group B, Group D and Group G as defined in  claim 2 .  
     
     
         11 . A gene controlling agent characterized in that the gene controlling agent comprises a compound possessing an action of decreasing an expression level of at least one gene selected from Group A, Group C, Group E and Group F as defined in  claim 2 , and/or increasing an expression level of at least one gene selected from Group B, Group D and Group G as defined in  claim 2 , in a living body to which stress is loaded.  
     
     
         12 . A gene controlling agent characterized in that the gene controlling agent comprises a compound possessing an action of decreasing an expression level of at least one gene selected from Group A and Group E as defined in  claim 2  in a living body to which stress is loaded, wherein the expression level of at least one gene is increased by the stress.  
     
     
         13 . A gene controlling agent characterized in that the gene controlling agent comprises a compound possessing an action of increasing an expression level of at least one gene selected from Group B and Group G as defined in  claim 2  in a living body to which stress is loaded, wherein the expression level of at least one gene is increased by the stress.  
     
     
         14 . A gene controlling agent characterized in that the gene controlling agent comprises a compound possessing an action of decreasing an expression level of at least one gene selected from Group C and Group F as defined in  claim 2  in a living body to which stress is loaded, wherein the expression level of at least one gene is decreased by the stress.  
     
     
         15 . A gene controlling agent characterized in that the gene controlling agent comprises a compound possessing an action of increasing an expression level of at least one gene selected from Group D as defined in  claim 2  in a living body to which stress is loaded, wherein the expression level of at least one gene is decreased by the stress.  
     
     
         16 . The gene controlling agent according to any one of  claims 10  to  15 , wherein the compound is a substance obtained by the method of any one of  claims 1  to  8 .  
     
     
         17 . The gene controlling agent according to any one of  claims 10  to  16 , wherein the gene control agent is purposed for mitigating or preventing stress.  
     
     
         18 . A method of controlling a gene characterized by administering to a living body either at least one compound selected from the group consisting of compounds of (i) and (ii) as defined in  claim 1  and a compound possessing an activity for controlling gene expression to substantially the same extent as that of said compounds for at least one gene, or a composition comprising the compound, thereby decreasing an expression level of at least one gene selected from Group A, Group C, Group E and Group F, and/or increasing an expression level of at least one gene selected from Group B, Group D and Group G, wherein Groups A to G are as defined in  claim 2 .  
     
     
         19 . A method of controlling a gene characterized by administering to a living body to which stress is loaded either at least one compound selected from the group consisting of compounds of (i) and (ii) as defined in  claim 1  and a compound possessing an activity for controlling gene expression to substantially the same extent as that of said compounds for at least one gene, or a composition comprising the compound, thereby decreasing an expression level of at least one gene selected from Group A, Group C, Group E and Group F, and/or increasing an expression level of at least one gene selected from Group B, Group D and Group G, wherein Groups A to G are as defined in  claim 2 .  
     
     
         20 . The method according to  claim 18  or  19 , wherein the compound possessing an activity for controlling gene expression to substantially the same extent as that of compounds of (i) and (ii) in  claim 1  for at least one gene is a substance obtained by the method of any one of  claims 1  to  8 .  
     
     
         21 . A substance obtainable by the method of any one of  claims 1  to  8 .  
     
     
         22 . The substance according to  claim 21 , which is a substance derived from a plant, a substance derived from Basidiomycetes, or a derivative thereof.  
     
     
         23 . The substance according to  claim 21  or  22 , which is a polysaccharide, a degradation product thereof, or a derivative thereof.